We have focused our studies in a number of different areas: 1. Treg control the size of the memory-phenotype T cell population. During the course of studies in the LCMV model, we attempted to use to explore the role of Treg by specifically depleting Treg with Diptheria Toxin (DT) using mice that express the DT receptor exclusively on Foxp3+ Treg (4, Foxp3-DTR mice). Administration of DT to these mice results in the transient depletion of > 95% of Foxp3+ Treg. Although these studies were not useful in the LCMV experiments, the use of these mice affords us with an opportunity to study the effects of polyclonal Treg in the steady state and to address some of the basic functions of Treg in the absence of inflammation or autoimmune disease. The Treg population was reconstituted within 72h following almost complete depletion when Foxp3-DTR mice were treated with DT. Treg reconstitution was mediated in part by IL-2, but the cellular source of the IL-2 remains to be determined. DT-induced Treg depletion in adult mice rapidly results in a three-fold expansion of activated memory phenotype CD44hiCD4+ T cells (Fig. 4). Expansion was completely unaffected by the administration of anti-MHC Class II. The increase in proliferative rate was observed in a model where true memory T cells could be assessed by MHC Class II tetramer staining following acute LCMV infection. Depletion of Tregs also resulted in the proliferation of nave T cells presumably in response to autoantigens, as proliferation was completely blocked by anti-MHC class II. 2.Role of cell surface and secreted TGF-&#946; in Treg Function. TGF-&#946; can have pleotropic effects on different cell types ranging from immune suppression, the promotion of fibrosis, to promotion/suppression of tumor growth. Since TGF-&#946; is expressed on the cell surface of Treg cells, one might assume it would be a major mediator of Treg suppression. Although this remains possible, our preliminary studies suggest that cell surface associated TGF-&#946; may have other unique functions in modulating immune responses. We have demonstrated that GARP or LRRC32 functions as a cell surface receptor for latent-TGF-&#946;. Using siRNA technology, we have shown in vitro that GARP+LAP- cells can bind latent TGF-&#946;, but we have not been able to determine whether in vivo the binding of latent TGF-&#946; to GARP occurs exclusively intracellularly or whether GARP can bind secreted latent TGF-&#946; or TGF-&#946; associated with LTBP. Although the GARP/LAP complex is expressed by platelets, within the immune system GARP/LAP expression is mostly observed on activated Foxp3+ Treg. The function of GARP/LAP on Tregs remains elusive. Our initial functional studies demonstrated that GARP/LAP contributed modestly to Treg-mediated suppression in vitro, but whether this holds in vivo remains unclear. To comprehensively examine the regulation of GARP/LAP expression, we generated a panel of mAbs to mouse GARP and have obtained mAbs to mouse LAP from Dr. H. Weiner, Harvard). In contrast to non-activated human Tregs, GARP is expressed at a low level on resting mouse Tregs, and is upregulated by TCR stimulation. GARP is also upregulated by stimulation with IL-2 or IL-4 alone, and to a lesser extent IL-7 or IL-9. Immediately following stimulation, GARP is expressed in the absence of LAP, but most cells co-express GARP and LAP within 48 h after stimulation. The GARP/LAP complex can be induced in vivo by administration of IL-2/anti-IL2 immune complexes. Both GARP and LAP are expressed on mouse iTregs generated in vitro and in vivo. 3. Role of GITR/GITR-L interactions in Treg function. Treg control immune homeostasis, but are also responsible for the suppression of tumor immunity; thus, reversing Treg function is a promising strategy for cancer therapy. The GITR is highly expressed on Treg, and upregulated on Tconv after activation. The GITR-L is primarily expressed on APC. Manipulation of GITR/GITR-L interactions in vivo has resulted in enhancement of immune responses, but whether the enhancement is secondary to co-stimulation of Tconv or reversal of Treg-mediated suppression remains unclear. To resolve this controversy, we used GITR-/- and GITR-L-/- mice and Fc-GITR-L in several experimental models. Treatment of mice with Fc-GITR-L induced the expansion of Treg. Polyclonal GITR-/- Treg survived less well than WT Treg following transfer to normal mice suggesting that GITR/GITR-L interactions are important for the survival of Tconv. In contrast, the expansion of both antigen-specific WT Tconv and Treg was not impaired following transfer to GITR-L-/- recipients. Although the capacity of GITR-/- Treg to suppress IBD was similar to WT, injection of GITR-L exacerbated IBD in mice that received CD45RBhigh T cells and in mice that received and CD45RBhigh cells and Treg. GITR-L treatment in this latter group resulted in a loss of Foxp3 expression. Thus, the GITR is a redundant molecule for costimulation of Tconv, but engagement of the GITR on Treg can result in different outcomes (expansion or loss of Foxp3) depending on the nature of the immune stimulus. Treg control immune homeostasis, but are also responsible for the suppression of tumor immunity; thus, reversing Treg function is a promising strategy for cancer therapy. The GITR is highly expressed on Treg, and upregulated on Tconv after activation. The GITR-L is primarily expressed on APC. Manipulation of GITR/GITR-L interactions in vivo has resulted in enhancement of immune responses, but whether the enhancement is secondary to co-stimulation of Tconv or reversal of Treg-mediated suppression remains unclear. To resolve this controversy, we used GITR-/- and GITR-L-/- mice and Fc-GITR-L in several experimental models. Treatment of mice with Fc-GITR-L induced the expansion of Treg. Polyclonal GITR-/- Treg survived less well than WT Treg following transfer to normal mice suggesting that GITR/GITR-L interactions are important for the survival of Tconv. In contrast, the expansion of both antigen-specific WT Tconv and Treg was not impaired following transfer to GITR-L-/- recipients. Although the capacity of GITR-/- Treg to suppress IBD was similar to WT, injection of GITR-L exacerbated IBD in mice that received CD45RBhigh T cells and in mice that received and CD45RBhigh cells and Treg. GITR-L treatment in this latter group resulted in a loss of Foxp3 expression. Thus, the GITR is a redundant molecule for costimulation of Tconv, but engagement of the GITR on Treg can result in different outcomes (expansion or loss of Foxp3) depending on the nature of the immune stimulus.